As more strains of human immunodeficiency type 1 (HIV-1), the virus that causes AIDS, become resistant to existing therapies, it is important to identify and characterize new targets for viral inhibition. The HIV-1 glycoprotein Env (gp120/gp41) is an attractive target because it mediates the initial stages of infection-viral attachment and membrane fusion. The interaction of Env with cellular receptors triggers conformational changes that ultimately lead to formation of a crucial trimer-of-hairpins structure in which the N- and C- terminal regions of the gp4 1 ectodomain associate. Peptides derived fiom the C-terminal region (C-peptides) inhibit membrane fusion by binding to the gp41 N-terminal region and preventing formation of the trimer-of- hairpins. Recently, we designed the 5-Helix protein to target the C-peptide region on the gp41 ectodomain and demonstrated potent, broad-spectrum inhibition of HIV-1 membrane fusion. While these studies have established the N- and C-terminal regions of the gp41 ectodomain as targets for HIV-1 entry inhibition, the physical bases underlying the activities of C-peptides and 5-Helix remain unclear. The studies proposed here are designed to explore the physical principles and mechanistic details of gp41 inhibition. The project's specific aims are: 1) To determine a high-resolution structure of 5-Helix protein using X-ray crystallography to validate the design strategy; 2) To characterize the interaction of 5-Helix and C-peptides by scanning mutagenesis and to correlate observed binding parameters with measured inhibitory potencies; 3) To map and characterize the determinants of resistance to 5-Helix and C-peptide inhibition; and 4) To determine the inhibitory stoichiometry of 5-Helix and C-peptides using functionally-complemented Env hetero-oligomers containing escape mutations obtained in Aim 3. These studies will explore whether 5-Helix and C-peptide inhibition is a thermodynamically or a kinetically driven process and how many molecules are needed to inhibit each gp41 trimer. The experiments will test susceptibilities of the two gp41 inhibitory epitopes to escape mutagenesis, and the results will provide insights into the structure and function of gp41 as it folds into a fusion-active conformation. This mechanistic information will be valuable for development of antiviral therapeutics and HIV- 1 vaccines that target the HIV- 1 gp4 1 ectodomain.